it include various food born diseases and include food born pathogen which caue various diseases to human.

1. Enumeration and detection of food borne pathogen in
food matrices and PCR based detection of Salmonella
MSc. Microbiology dissertation work
School of life science, JNU, Jaipur
July 2017
Under supervision of
Dr. K Neelakanteshwar patil
Senior scientist@
CSIR-CFTRI, Mysuru
Submitted by:
Irshad Khan
M.Sc. Microbiology
JNU, Jaipur

2. Introduction
Food- food are the things which
we ate and we get optimum
amount of energy and nutrient
necessary for our survival.
 Food contamination
 Food born diseases
 Food safety

3. http://slideplayer.com/slide/6360370/

5. OBJECTIVE OF THE STUDY
In this part of study we aim to understand various
microbiological parameters for enumeration, detection and
identification for raw and processed foods.
Enumeration parameters that include Total Plate Count, Coliform
count and yeast & mold count
Enumeration of pathogens such as Escherichia coli, Staphylococcus
aureus and Bacillus cereus.
Detection of Pseudomonas aeruginosa, Salmonella species, Listeria
monocytogenes and Shigella species.
Microbial Evaluation of canned foods of commercial marketed
sample

6. MATERIAL AND METHOD
There are various steps for enumeration and detection of food born
diseases. From basic microbiological plating to standard
biochemical testing.
I have used mainly
1. selection media and sample preparation
2. selective and non selective growth
3. Biochemical testing
4. Standard method of Biochemical confirmation using Kit
5. Colony PCR especially for Salmonella confirmation

7. Results and Discussion

8. Enumeration of Yeast and mold count
Matrix A = 63+65/2×102
64×102CFU/g
Matrix B = Not detected (<10 CFU/g)
Yeast and mold growth is seen there
in Chilli powder .
It means that spicy food have greater
risk of yeast and mold contamination
then wheat powder
A-Chilli
B-Wheat Atta

9. The colonies were observed microscopically (40X) by staining with lacto
phenol cotton blue. The hyphae and conidial structure resembled the
genus Cladosporium.
Morphological identification of mold

10. Enumeration of Coliform Count
A-Skim milk powder
B-Wheat Atta
Matrix A = 124+126/2×101
125×101CFU/g
Matrix B = Not detected (<10 CFU/g)
Coliform growth is seen there in Skim
milk powder.
skim milk powder is at greater risk of
coliform contamination

11. Enumeration of Total Aerobic mesophilic count
wheat Atta was microbiologically safer than skim milk powder. And also the
bacterial count of skim milk powder is exceeding the FSSAI permissible limits
(>2,50,000 CFU/g) rendering it unsafe for consumption
A- Wheat Atta
B- Skim Milk
Matrix A
CFU/g = 230+236/2×104
=233×104CFU/g
Matrix B
CFU/g = 35+33/2×102
=34×102CFU/g

12. Detection of Salmonella Species using Selective media
Presence of presumptive Salmonella colonies were observed on XLD, HEA
and BSA of matrix A. the colony morphology these selective agar.

13. Observation of TSI and LIA for matrix A and B; shows that matrix A is
positive for Salmonella.
Primary biochemical test for Salmonella species
Typical colonies of Salmonella Species picked up from Selective agar plate For Biochemical
test using TSI and LIA slants.

14. Biochemical confirmation test for Salmonella
Species using Enteriobactria Kit
Results with Enteriobactria Kit confirmed that, there is Presence of
Salmonella Species contamination in Wheat Atta

15. typical colonies on MacConkey agar plate were observed in matrix A and
no colonies were found in matrix B. colonies appear as
translucent/slightly pink in color with rough.
There might be chances of presence of Shigella Species
A- Wheat Flour
B- Milk
Detection of Shigella Species

16. Result of TSI confirmed that yes there is greater chances of Shigella Species
presence in given wheat flour
Biochemical test for Shigella on TSI slant
Typical colony were inoculated into TSI agar Slat

17. Biochemical confirmation test for Shigella Species
using Enteriobactria Kit
Results with Enteriobactria Kit confirmed that, there is Presence of
Shigella Species contamination in Wheat flour

18. Detection of Listeria monocytogenes Species
A- Rice bath
B- Chilli
powder
Typical colonies on oxford agar plate of matrix A appeared black with a black halo.
showing Listeria monocytogenes typical colonies

19. Confirmation of Listeria monocytogenes by motility and Catalase test
In Sample A there is tumbling mobility in seen .
Catalase test result is positive for Sample A.
Results of Motility test and Catalase test confirmed that there is Listeria
Monocytogenes contamination in Sample A (Rice Bath)
1- Positive
2- Negative
1- Positive
2- Negative

20. Enumeration of Bacillus cereus
For the growth of Bacillus cereus, samples were spread on Mannitol Yolk
Polymyxin (MYP) Agar plate
appeared eosin pink colonies surrounded by lecithinase zone in Biscuit and No
colonies were found in Cake
showing bacillus Cereus typical colonies are present in Biscuit
A- Biscuit
B- Cake

21. Confirmation of Bacillus Cereus using gram staining;
fermentation of dextrose and Nitrate Reduction test
Gram staining Nitrate Reduction
Dextrose Fermentation
Yellow color and Gas formation was observed
Bacillus cereus were confirmed by the result of Gram staining; Fermentation of
dextrose and Nitrate Reeducation test

22. Detection of Pseudomonas aeruginosa in water sample
Pseudomonas aeruginosa was detected in water sample. APB with membrane filter
paper containing microbial load of water observed in fluorescent which gave the
green fluorescence that was compared to standered (MTCC9543)
Growth in Asparagine prorine broth (APB)- Fluorescent Assay-

23. Confirmation of Pseudomonas aeruginosa
Typical colonies were found on produces a yellowish to green diffusible
pigment. Positive colonies of Pseudomonas aeruginosa gave florescence.
Casein Hydrolysis on Milk agar plate Fluorescent assay

24. Detection and Enumeration of Staphylococcus aureus
Given samples were spread on Baired Parker Agar plate.
Colonies of S. aureus on baird parker agar appeared black, circular,
smooth, convex, 2-3 mm diameter and sticky when touched.
This Suggest that Cake sample have contamination of S. aureus
A- Cake
B- Biscuit
Colonies on S. aureus on BP agar plate

25. Confirmation Test for Staphylococcus aureus
Coagulase test and Catalase test result confirmed that Staphylococcus
aureus is present in given Sample A

26. Enumeration of S. aureus
GC( giolitti cantoni) broth tube
Blackening of the medium are indicates the presence of Staphylococcus aureus.
Tubes 1 and 2 (10-1 dilution) 3 & 4 (10-2dilution) are showing positive result for
Staphylococcus aureus and 5 or 6 (10-3 dilution) showing negative.

27. Enumeration and detection of E.Coli
Inoculated given water sample in LST- broth for E. coli enumeration in water sample in
different volume

28. Florescence Assay of given water sample inoculation primarily suspect that there
is presence of coliform in water sample .
Florescence Assay for primary Analysis of given
Water Sample

29. Confirmation test for coliform
Gas production is observed . Hence Coliform confirmed in suspected water
sample.

30. EMB plate typical nucleated dark centered colonies with or without
metallic sheen.
Direct observation of EMB plate for E.Coli by streaking

31. Observation of E.Coli by Gram Staining under microscope
Gram negative non-spore forming rods was seen.

32. Confirmation of E.Coli by typical biochemical IMViC test
1- Positive
2- Negative
1-Negative
2- Positive
1-Negative
2- Positive
1-Negative
2- Positive
Results of IMViC test confirmed that there is typical E. coli is present in
given water sample

33. Examination of Canned food sample for microbial confirmation
Direct observation of Canned food
packet
1- Positive
2- Negative
1- Negative
2- Positive
By direct observation of packet, growth in CMM and growth in DTB
confirmed that given canned food sample is contaminated

34. PCR based confirmation of Salmonella species from colony
Results of colony PCR from Salmonella species specific agar plate
confirmed that Salmonella is present on given agar plate

35. CONCLUSION
The Dissertation work involves the evaluation of microbial parameter for hygiene and safety as well as detection of
food borne pathogens. The safety parameters include Total plate count, Coliform count, yeast and mould count, S.
aureus count. The detection of few food borne pathogens as per US FDA was also conducted as a part of the study
includes Salmonella, Shigella, Pseudomonas aeruginosa, Bacillus cereus, Listeria monocytogenes. The detection of these
pathogens was performed as per the conventional analysis with standard protocols from the [Compendium of methods
for the microbial examination of foods] APHA, 2015
In addition molecular approach (PCR based) for detection of Salmonella was also a part of this study, where
molecular approaches are gaining more importance in recent time that could be a better replacement for the time
consuming convention detection protocol.
Microbial evolution of canned foods which is of major concern today was also considered as a part of the study.
Hence, the study is in detail of the 12 parameters that are key for a microbial analysis of the food approach.

36. Refrences
 Bhattacharya, Seshadri Sekhar,Choudhury, Basanta kumar, 11/09/2010. Studies on two serotypes of salmonella isolated from blood samples of patients of an industrial hospital.
 Divyashree, M. S., shamala, T.R., 2008. Polyhydroxyalkanoate from Bacillus sp its production isolation and characterization, http://hdl.handle.net/10603/38487.
 Khan, javed ahamad, 2014. Enumeration, detection and characterization of food borne bacterial pathogens by conventional and molecular techniques
 Hens, Dipak Kumar, Kumar, Ranajit and Chatterjee, 2005 Study of drug resistance and virulence factors among Shigella species.
 Mandappa, I. M., Manonmani, H. K., 2014. Molecular detection of Bacillus cereus and its toxins in food, http://hdl.handle.net/10603/71681
 Parekh Tarak, n.d. 2009. Detection of food borne pathogens by molecular methods and use of molecular methods in foods
 Srividya, Y, 2012. Murali, H. S., Molecular detection of listeria monocytogenes,
http://hdl.handle.net/10603/74886
 Varghese, Siju M, 2015 Integron study, molecular typing and characterization of salmonella serotypes isolated from seafood and poultry. Cochin University of
Science and Technology.
 Web References
 National central and disease control December, 2009, vol.13 no. 14. (http://www.ncdc.gov.in/writereaddata/linkimages/Dec_091047732317.pdf . Accessed 2017
May 02
 World Health Organization, 2006. Chapter 9, page No-148. http://www.who.int/water_sanitation_health/hygiene/emergencies/em2002chap9.pdf . Accessed 2017
May 05
 Role of microbes in food processing, Biology articles December 2015, class 11. http://byjus.com/biology/role-of-microbes-in-food-processing/ .
 Alexander Gaffney RAC, June, 2014. Regulation US FDA, http://www.raps.org/regulatory-focus/news/2014/06/19395/FDA-Final-Rule-On-Qualifying-GAIN-Act-
Pathogens/ . Accessed 2017 May 05
 Food safety and standards authority of India, Regulation July, 2011. http://fssai.gov.in/home/food-standards/fss-regulations.html
 Business maps of food Industry India July, 2015. http://byjus.com/biology/role-of-microbes-in-food-processing/
 Dilek keskin, sanver ekmekcl 2015. Investigation of the incidence of pseudomonas Spp. In food.
http://www.hjbc.hacettepe.edu.tr/site/assets/files/3386/35_3_181-186.pdf

37. Words of thanks
o First of all I want to thanks my Parents and family for there continuous healp and support to me
for encouragement for Study, and so finally today I am going to completing my Masters degree.
o Thanks to chairmen and administration of Jaipur National college, (Jagatpura) for there
supportive help for offering M.Sc. Microbiological programme.
o Thanks to H.O.D. of Life science department, vice chanceller, and coordinator of M.Sc.
Microbiology programme.
o I would like to thanks my institutional guide for Masters dissertation work Ms. Upasana Rishiraj
for completion.
o Special thanks to Senior Scientist- Dr. K NEELAKANTESHWAR PATIL and all lab members at CSIR-
CFTRI, Mysuru.
o I am thanks to all my batch mats for there continues support during the period of my masters
programme
o Finally I would like to say special thanks to my Best friend Praveen Kumar Verma (IISc,
Bangalore) for his continuous support for encouraging me for studies from my bachelor's
programme and helping me in completion of dissertation work.